Second-order calibration serves as a remedial measure for the simultaneous determination of andrographolide and dehydroandrographolide in Andrographis paniculata and its preparations by HPLC without complete baseline separation.

In this work, high-performance liquid chromatography with diode array detection was applied for the simultaneous determination of andrographolide and dehydroandrographolide in Andrographis paniculata and its preparations. As a result of the incomplete baseline separation caused by complex backgrounds, the classical univariate calibration method failed to determine accurate contents of the analytes. On this occasion, chemometric second-order calibration based on the well-known alternating trilinear decomposition algorithm was then explored to serve as a post-experimental remedial tool to solve this problem. By using the intelligent "mathematical separation" of alternating trilinear decomposition, the peak areas of the analytes do not need to be directly measured and the predictive results become accurate. The contents of andrographolide and dehydroandrographolide were determined to be (7.95 ± 0.15) and (1.85 ± 0.02) μg/mL for Andrographis paniculata, (1.34 ± 0.01) and (5.53 ± 0.04) μg/mL for its preparations, which was in agreement with those obtained by a reference liquid chromatography with mass spectrometry method. This study showed the superiority of second-order calibration method over classical univariate calibration method for simultaneous determination of multi-analytes in complex samples. It also proved that second-order calibration may be a good choice for remedying incomplete baseline separation problem, with the accompanied reduction of experimental burden and toxic organic solvents as well as analysis time and cost.